Following a vascular injury or during inflammation, endothelial cells rapidly release the contents of their storage granules, called Weibel- Palade bodies (WPBs). The major soluble component of these granules is von Willebrand factor (vWf). vWf is a large multimeric glycoprotein that has a dual role in hemostasis: it promotes platelet adhesion and it protects factor VIII (FVIII) against proteolysis. vWf is synthesized with a prosequence which directs multimer formation through an as yet unknown mechanism. The formation of the WPBs and the function of vWf after secretion are the main topics of this proposal. We will be greatly assisted by the existence of vWf-deficient mice that we prepared during the last grant period. The proposal has four specific aims. I) Weibel-Palade body formation. We will examine whether the vWf prosequence has a disulfide isomerase enzymatic activity, which would explain its role in vWf multimerization. We will continue our studies on the molecular mechanisms involved in granulogenesis. II) Role of vWf in normal physiology. We will evaluate the effect of vWf-deficiency of FVIII half life and secretion. We will examine whether there is a feedback mechanism by which vWf or its prosequence modulates FVIII biosynthesis. We will study the role of vWf in platelet thrombus formation in vitro and in vivo using a new intravital microscopy model we have developed. The importance of vWf will be compared with that of other platelet adhesion molecules, i.e., fibrinogen and beta3 integrins. III) Role of vWf in various diseases. We will test the role of vWf in diseases in which platelets are thought to play an important part, e.g., septic shock, stroke and atherosclerosis. IV) Generation and characterization of new mutant mice. We will produce combinations of genetic defects by crossing the vWf-deficient mice with other mutant strains. We will evaluate the effect of gene dosage of vWf in homeostasis and disease by producing mice with twice the normal level of vWf. Our research will employ a variety of techniques including protein chemistry, cell culture, intravital microscopy, animal experimentation, and finally, genetic engineering of a targeted gene duplication in mice. Learning about the mechanisms involved in WPB formation and secretion is clinically relevant, as the main treatment of patients with low vWf relies on releasing the stored protein pool. An understanding of the exact roles that vWf plays in platelet adhesion and thrombus formation may lead to new anti-thrombotic therapies which could become applicable in diseases where platelet adhesion and/or aggregation is part of the disease process.